The MRamp strategy was initially designed with the goal of improving the molecular sensitivity of MR imaging by modulating the MR signal output on two levels simultaneously: 1) target specificity: a pair of receptor-targeted enzymes co-localize in the specific tissue compartment and due to enzymatic activity complementation, enable rapid modification of low molecular weight paramagnetic substrates which results in their local retention at the reaction site; and 2) sensitivity: local retention gives rise to an amplified MR signal generated by both high local concentration density and increased relaxivity of the paramagnetic products of the enzymatic reaction. We have marked major milestones in bringing the MRamp technique closer to clinical translation including: 1) demonstration of MRI of endogenous myeloperoxidase activity in human pathology and in various disease models; 2) successful imaging of receptor expression in cancer models. MRamp is one of the few existing techniques enabling the detection of the co-expression of two protein markers (receptors). We recently explored co-expression imaging feasibility with PET-CT in addition to MRI. By implementing an expanded multi-modality imaging approach (BLI, MRI and PET-CT), we previously identified osteolytic and non-osteolytic triple-negative breast cancer (TNBC) phenotypes and characterized them using the analysis of key gene transcripts. The new phase of research proposed here, will include the optimization of a dual receptor imaging strategy and its application to in vivo imaging of two critically important biomarkers of TNBC, i.e. overexpressed EGFR and PD-L1. The ability to image the co-expression of these critical markers in TNBC tumors is expected to provide a new, highly quantifiable and easily interpretable diagnostic imaging method that can be used to (1) detect metastatic spread, (2) select patients for targeted therapies and (3) monitor therapeutic response or resistance over the course of treatment. In addition to simplifying the monitoring of patients undergoing existing cancer treatments, this new methodology could speed testing of investigational new drugs in clinical trials thus expediting approval of promising new TNBC therapies. Our initial goal will be to screen novel chimeric small molecule catalyst-substrates, to select candidates for highest MR and PET signal amplification using our enzyme dependent imaging system. We have previously published on inroads made in our design of MR detectable paramagnetic Mn(II)-based superoxide dismutase mimetics and will further harness their power as tools for miniaturizing our MRamp system. Such miniaturization may prove essential for the ability to tag and track extracellular vesicles (EV) in vivo which are now believed to be a key element in the metastatic spread of cancers such as TNBC. Therefore, a second major goal of this proposal will be to image endogenously occurring EVs using our dual receptor, high sensitivity enzyme complementation approach.